PROJECT SUMMARY/ABSTRACT Preeclampsia (PE) is a common pregnancy disorder and a leading cause of maternal and perinatal death. While PE affects up to 10% of pregnancies, predictive diagnostics and therapies remain extremely limited, as PE likely arises from multiple etiologies that converge to present the same general symptoms. Adaptive immunity has been implicated in a subset of PE; however, its specific role in the pathophysiology of PE remains unclear. Thus, it is essential to further investigate adaptive immunity's role in PE to inform the development of interventions needed to improve short- and long-term outcomes for both mother and baby. The sole known cure for PE is removal of the placenta, the likely causative organ. Despite this, many immunologic PE studies have merely tested peripheral blood. Our unique approach allows us to determine the role of adaptive immunity in PE comprehensively: from an already-established collaboration we acquire maternal peripheral blood, umbilical cord blood, and placental tissue from normal pregnancy (NP) and PE donors. Clinical experts will select for PE cases that implicate immunity. Our objectives are two-fold: First, we want to test if Treg suppressive mechanisms are altered during PE. Second, inflammation can cause potentially-aberrant, antigen-nonspecific immune responses (termed bystander activation). Because PE is characterized by exaggerated inflammatory responses, we want to determine if this heightened inflammation is sufficient to bystander active antigen-nonspecific CD8+ T cells and render them capable of killing in an innate-like manner. To achieve these objectives, our lab has developed and validated multiple 28-color fluorescence cytometry assays, which interrogate cell phenotype, activation, and functional capacity (i.e. expression of exhaustion markers, inhibitory receptors, co-stimulatory molecules). We will employ these assays to determine the composition and capabilities of cells directly ex vivo. Additionally, we will conduct in vitro co-cultures and stimulations to ascertain if bystander-mediated killing, Treg-mediated suppression, and the production of anti- and pro-inflammatory cytokines is altered during PE. Hypothesis: Our central hypothesis is that dysregulated adaptive immunity drives severe PE, specifically: 1. Regulatory axes of adaptive immune cells are perturbed during PE, impairing Treg suppressive function while promoting the accumulation of highly pro-inflammatory cells systemically (and most profoundly at the placenta). 2. Dysregulated inflammation in PE permits aberrant cellular behavior (i.e. bystander activation) in adaptive immune cells, which further contributes to PE pathophysiology through their innate-like killing mechanisms. We will test these hypotheses through two independent aims: Aim 1: Characterize Treg phenotype and suppressive function from PE and NP tissues. Aim 2: Test the ex vivo cytotoxic potential of antigen-nonspecific cells during NP and PE, and if they can kill target cells using innate-like mechanisms. Our proposed experiments will elucidate the role of adaptive immunity in preeclampsia, which in alignment with the mission of the NIH, may inform development of interventions to improve maternal and perinatal health.